To genetically manipulate key proteins involved in the autonomic regulation process, we have developed a technique for the culture of rabbit sinoatrial node cells, as it is impossible to do so in freshly isolated SANC. We have been able to obtain stable adult rabbit cultured SANC (c-SANC) to characterize their properties, and have successfully overexpressed proteins in c-SANC via adenovirus-directed acute gene-transfer technique. Our results show that on the first day of primary SANC culture, most of the cells tend to spread out and could stay alive for up to 8 days. By immunostaining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 ryanodine receptors (RyR2), L-type Ca2+ channel, hyperpolarization-activated cyclic nucleotide-gated channel 4, phospholamban (PLB), Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a and Sodium-Calcium exchanger. At 34 plus/minus 0.5 degrees C, c-SANC generate spontaneous, rhythmic action potentials (APs), but at a level (1.35 plus/minus 0.02 Hz, n=804, over 2 to 8 days into culture) roughly 50% of that of f-SANC (2.79 plus/minus 0.04 Hz, n=203, p<0.001). Although both c- and f-SANC generate rhythmic APs, the rhythmicity of c-SANC AP beating interval (APBI) is less robust than that of f-SANC, as indicated by a lower rhythmicity index of the autocorrelation function, a lower power spectrum amplitude and a bigger coefficient of variation (CV) in c-SANC versus f-SANC (p<0.001). The CVs of classic and additional analysis of AP characteristics are also increased in c-SANC vs. f-SANC. Spontaneous Local Ca2+ Releases (LCR) period are increased in c-SANC, and are correlated with the beating intervals of AP triggered global Ca2+ release transients the decay time of Ca2+ transients in both cell types, but with an increased CV in c-SANC vs. f-SANC. The reduced rhythmicity in c-SANC APBI is associated with prolongation of spontaneous LCR period during diastolic depolarization and an increase in its coefficient of variation (0.199 plus/minus 0.014 (n=41) for c-SANC vs. 0.122 plus/minus 0.009 (n=32) for f-SANC, p<0.001). It is well documented that the peptide inhibitor of protein kinase A (PKA), PKI, can dramatically reduce or stop the beating rate of f-SANC. We hypothesized that the low beating rate of c-SANC is due to the down-regulated PKA signaling in the cultured cells. Indeed, non-specific PDE inhibitor IBMX (100 microMolar, 10min) increases the AP firing rate of c-SANC to a similar maximum to the treatment of f-SANC. Furthermore, acute stimulation of beta-adrenergic receptors with 1 microMolar isoproterenol (ISO) for 10 min accelerates AP and Ca2+-transient kinetics, reduces the LCR period and accelerates the AP firing rate to a similar maximum in c-SANC (3.34 plus/minus 0.05 Hz, n=150) and f-SANC (3.55 plus/minus 0.06 Hz, n=126). In addition, we observed that the phosphorylation level of RyR2, indexed by the fluorescence density of phosphorylated RyR2 at Ser2809 normalized by total RyR2 fluorescence density, is substantially lower in c-SANC (1.32 plus/minus 0.06, n=47) than in f-SANC (1.66 plus/minus 0.15, n=24, p<0.01). While acute ISO stimulation raises the RyR2 phosphorylaiton at Ser2809 to a similar level in both cell types, PKI treatment reduces the phosphorylation level. More specifically, the phosphorylation level of PLB at Ser16, a PKA specific site, is also significantly lower in c-SANC than f-SANC. What is the mechanism underlying the PKA down-regulation in cultured pacemaker cells? Based upon the fact that the activation of pertussis toxin (PTX)-sensitive Gi signaling is involved in the beating rate reduction of f-SANC, we measured the protein expression level of type 2 regulator of G protein signaling (RGS2), which functions as a powerful negative regulator of PTX-sensitive Gi signaling. As we expected, the protein level, indexed by the immunolabeling density along the cell membrane, is substantially lower in 2 day cultured SANC (149.9 plus/minus 4.0, n=100) than in f-SANC (201.9 plus/minus 6.0, n=88, p<0.001). 2 hours incubation of 1 microMolar ISO enhances the staining density of RGS2 and PKI completely inhibits ISOs effect. Functionally, overexpression of RGS2 via adenovirus-directed acute gene-transfer technique increases the spontaneous beating rate of cultured SANC from 1.35 plus/minus 0.05 Hz (n=91) to 1.86 plus/minus 0.05 Hz (n=50, p<0.001), which is 66% of f-SANCs AP firing ate. This effect is not because of adenovirus infection, as introducing the green fluorescent protein (GFP) into c-SANC via the same technique, does not affect the cell beating rate. Furthermore, when cultured SANC were treated with 0.4micrograms/ml PTX overnight, the spontaneous beating rate is boosted to 2.38 plus/minus 0.11 Hz (n=45), 85% of f-SANCs AP firing rate. Partial rescue of c-SANCs AP firing rate by PTX treatment or RGS2 overexpression indicates that a reduction in PKA-dependent Ca2+-cycling protein phosphorylation that is Gi-dependent is involved in prolongation of LCR period and reduced spontaneous AP firing rate of c-SANC, and that this deficit can be reversed by pharmacologic or genetic manipulation. Early studies in single SANC indicate that intrinsic pacemaker stimulation not only change the average action APBI but also determine APBI variability. We tested the idea that the extent of spatiotemporal synchronization of both spontaneous local diastolic RyR activation induced by phosphorylation of Ca2+ cycling proteins in intact SANC are determinants of both APBI and APBIV. We observed that reduced phosphorylation of SR Ca2+ cycling protein phosphorylation and increased average APBI that are characteristic of SANC in culture (c-SANC) were accompanied by spatiotemporal de-synchronization of spontaneous local RyR activation, manifest not only as a reduced average LCR size and increased average LCR period, but also in increased variability of LCR periods and increased variability of diastolic depolarization parameters that is modulated by diastolic LCR occurrence. beta-adrenergic receptor stimulation increased protein phosphorylation and reduced the average APBI in both c-SANC and f-SANC, increased the spatiotemporal synchronization of LCR periods and sizes, reduced the variability of diastolic depolarization parameters and reduced CV. Detailed analysis was also applied to action potential parameters in f-SANC with interventions increasing the APBI, e.g. PKI or Cch, and the variabilities were also increased. Thus, both the spontaneous AP firing rhythm and average AP firing rate of isolated SANC are linked to the extent to which random local spontaneous RyR activation become synchronized by the APBI and by SR Ca2+ cycling protein phosphorylation. The c-SANC model is a helpful tool. For example, overexpression of R299Q-AMPK gama2 via adenovirus (together with mCherry and conjugated Flag) acute infection in c-SANC, significantly reduce the spontaneous beating rate to half of no virus control, mCherry adenovirus control or wild type-AMPK gama2 (together with mCherry and conjugated Flag) groups, via the contraction measurement of transmission line scan images (p<0.001 via one-way ANOVA, n=2936 for group). Within each group, there is no correlation between the mCherry density and spontaneous beating rate. mCherry is the fluorescent marker guiding the choose of spontaneous beating cells with adenovirus infection, and there is positive correlation between mCherry and the Flag density of immuno staining.